FR2747044A1 - USE OF POST-GERMINATING VEGETABLE SEEDS AS A SOURCE OF SUPEROXIDE DISMUTASE AND COSMETIC, PHARMACEUTICAL OR AGRO-FOOD COMPOSITIONS CONTAINING SUCH SUPEROXIDE DISMUTASE VEGETABLE, AND METHOD OF EXTRACTING - Google Patents

Abstract

A superoxide dismutase, or SOD, is disclosed. Germinated plant seeds are used in accordance with the invention as a source of superoxide dismutase which can thereafter be complexed with a peroxidase and a enzyme cofactor. Cosmetic, pharmaceutical or agri-food compositions having anti-free radical activity can thus be obtained.

Description

 The present invention relates to a process for extracting / purifying a superoxide dismutase from seeds of plants after germination, and its combination with a peroxidase accompanied by its enzymatic cofactor. This stable enzyme complex in a cosmetic formulation can be used in cosmetic, pharmaceutical or food-processing compositions.

INTRODUCTION
More specifically, the invention aims to obtain a superoxide dismutase (abbreviated hereinafter SOD) from seed of germinated plants, that is to say having undergone a germination step, which has a strong enzymatic activity, and greater stability when this SOD prepared after germination of plant seeds is used in antiradical compositions, in combination with another enzyme capable of destroying the by-products of the superoxide disproportionation reaction, mainly hydrogen peroxide yes202) , such as catalases or peroxidases. The invention thus has an advantageous application in cosmetic compositions, pharmaceutical or dermatological compositions, or agri-food compositions.

 Free radicals are known to be atoms or molecules that have an unpaired electron on their outer orbitals. These are extremely unstable compounds that can react with the most stable molecules to match their electron.

 Oxygen free radicals are formed continuously in the body, in mitochondria or during phagocytosis processes. Physical factors such as exposure to ultraviolet light and the environment in which humans evolve are also factors of high production of free radicals in biological compounds. These factors are, for example, automobile pollution, tobacco, ionizing radiation, etc.

 Oxygen free radicals are formed by partial reduction of molecular oxygen. The capture of an electron by oxygen, will generate the superoxide radical o2A. This anion is not very reactive but it can generate very reactive radical species. The enzymatic disproportionation of the superoxide anion carried out by SOD leads to the formation of hydrogen peroxide which, in the presence of ferrous iron, undergoes the Fenton reaction to form the hydroxyl radical OH-, which is very reactive.

 Because of their high reactivity, free radicals are able to attack all cellular constituents and cause serious alterations: in the skin, free radicals, especially the superoxide anion, have the major target collagen but also elastin fibers, glycosaminoglycans and proteoglycans, intracellular DNA, phospholipids of cell membranes, etc.

 Collagen and elastic fibers directly capture the free radicals formed, at the cost of various degradations: chain breakage and release of small peptides degraded by nonspecific proteases, interchain bonds reducing the elasticity.

 Also, in order to cope with the destructive effects of activated forms of oxygen, it has become increasingly necessary to prepare compositions based on antioxidants in all fields, and in particular in cosmetics, pharmacy or food industry. . In cosmetics, such compositions have been placed on the market and are called antiaging or anti-aging products.

STATE OF THE ART
The antioxidants currently used in cosmetics or pharmaceuticals are either chemical molecules that are free radical scavengers or enzymatic systems whose major disadvantage is very poor stability even at room temperature.

 The lipophilic sensors used are generally vitamin E and carotene, components of cell membranes that protect them from lipid peroxidation by reducing the peroxide radicals formed.

 The hydrophilic antioxidants used generally consist of vitamin C, which acts in aqueous media with respect to superoxide and hydroxyl radicals, but also by enzymatic cofactors such as glutathione and zinc. Glutathione is the co-factor for many enzymes involved in antiradical defense (such as glutathione peroxidase, glutathione transferase, etc.) while zinc is the cofactor of SOD in cuivrezinc.

The enzyme used in cosmetics or pharmacy is SOD whose role is to ensure the almost instantaneous destruction of superoxide radicals potentially harmful to the body and skin tissues. The reaction catalyzed by SOD is the disproportionation of the superoxide anion into hydrogen peroxide or hydrogen peroxide according to the following chemical reaction:

There are several enzymes also known as superoxide dismutase isozymes which have been mainly isolated from wheat germ, and those skilled in the art will be able to refer to the Beauchamp article published in the review.
Biochimica and Biophysica Acta, 317 (1973), 50-64. In this article, Beauchamp reports having isolated three SOD enzymes, the so-called manganese-sensitive SOD Mn, which is not inhibited by 0.2mM cyanide but is inactivated by the treatment of Tsuchihashi based on chloroform plus ethanol. than two other SOD Cu-Zn SODs which are inhibited by cyanide but unaffected by treatment with chloroform plus ethanol. The latter contain a cupric ion Cu2 + and a Zn2 + ion per protomer and have a molecular weight of the order of 30000 daltons.

 It will be recalled that SOD Cu-Zn, present in the cutaneous tissues, constitutes the first line of enzymatic defense against the free radicals generated by the ultraviolet rays. But it is interesting to note that the enzyme sees its detoxification potential decrease sharply after irradiation with ultraviolet. This finding has, moreover, led the cosmetology or pharmacy professionals to incorporate SOD into formulations in order to reduce the level of free radicals present in the cutaneous tissues.

fi was thus proposed by L'Oréal in document
US-A-4,129,644 to use a superoxide dismutase enzyme in a cosmetic composition and in a method comprising applications for maintaining the keratin structure of the hair or for protecting the skin and the hair by applying such a SOD. SOD was mainly obtained from beef blood or different bacterial strains (claims 2 to 5).

 The L'Oréal company has further proposed in WO 92/19224 a topical anti-free radical composition based on SOD of various origins (animal, human, bacterial, yeast or biotechnological) and a phosphonic acid derivative as metal complexing agent to fight against skin aging and the protection of the skin against irradiation based on the demonstration that certain metal-inactivating complexing agents could, in certain cases, attenuate the production of hydroxyl radicals (OH ') toxic.

 It is still known from FR-A-2,634,125 Nippon a stabilized superoxide dismutase composition comprising SOD, phosphate, alkali metal chloride and sucrose (see the title claims). SOD is actually extracted from human blood.

FR-A-2 693208 Inocosm further discloses a process for obtaining an enzymatic composition of SOD of plant origin from cereals such as wheat germ by extraction with the aid of a liquid alcohol, removal of polyphenols with a fixing agent such as polyamide or polyvinylpyrrolidone, washing and finally extraction of proteins and enzymes again with liquid alcohol. The SOD activity obtained is very low and the use of solvents makes the SOD extract difficult to use.
So far, industrial SOD has been obtained by extraction from bovine erythrocytes available in large quantities in slaughterhouses.

On the other hand, although it is known since at least the articule of
Beauchamp et al. in Biochimica and Biophysica Acta, 317 (1973) pages 50-64, to extract SOD from wheat germ, and that at least one similar patent was deposited by Inocosm in 1992 (FR-A-2,693,208). ), the proportion of extraction
SOD of plant origin has been found to be very low due to a low proportion of SOD in the seeds of cereals and, secondly, by the use of a solvent extraction process low yield.

 Thus, the main object of the present invention is to solve the new technical problem consisting in the provision of a solution which makes it possible to obtain a plant-based SOD in large quantity with a very good yield so as to use it for the first time. industrial scale, particularly in the cosmetics, pharmaceutical or agri-food sector.

 It is another object of the present invention to solve the new technical problem of providing a solution which makes it possible to obtain a SOD of extremely active and also extremely stable plant origin, preferably being able to retain 80% of activity for 1 month (35 days) at room temperature and an activity of the order of 50% at 45 ° C., thus allowing efficient incorporation into anti-free radical compositions, in particular in cosmetic, pharmaceutical or food-processing compositions.

 All of these technical problems have been solved for the first time in a simple, safe and reliable manner by the present invention, which is an unexpected technical result and not obvious to one skilled in the art.

 Thus, according to a first aspect, the present invention relates to the use of seeds of plants after germination as a source of SOD extraction. In the context of the invention, the terms grains or seeds are equivalent.

 According to a particular embodiment, these plant seeds are cereal seeds or seeds of leguminous plants or seeds of oleaginous plants. As cereals, all cereals, especially rye, maize, wheat or barley, preferably barley, can be used among the varieties of barley that can be used, spring or winter varieties can be used . For legumes, seeds of any leguminous plant, but preferably lentil or pea seeds, will be used. For oleaginous plants, seeds of any oleaginous plant, but preferably soya beans, will be used.

 According to another variant embodiment, the preliminary germination step comprises germination for a period of time sufficient to increase the SOD activity. This period of germination time is variable according cereals and legumes but is easy to determine by the skilled person once it knows according to the invention that germination can increase the SOD activity.

 According to a presently preferred embodiment, the seed is germinated for several days in suspension in water, preferably in the presence of a germination promoting agent at room temperature. Such a germination promoting agent is preferably a compound belonging to the gibberellin family.

 According to a more preferred embodiment, germination of the seed is preceded by soaking in an aqueous solution at room temperature or cold for one or more days, for example two days. The initiation of the germination step is preferably carried out by adding a germination promoting agent, preferably a compound belonging to the gibberellin family.

 Other features of the use according to the invention will also become apparent from the claims and the description taken as a whole.

 According to a second aspect, the present invention also relates to a process for extracting SOD characterized in that one uses as a source seeds of plants having undergone a germination step.

 According to an advantageous embodiment, the germination step comprises suspending the seeds of plants in an aqueous solution for several days at room temperature.

 According to a preferred embodiment, this germination step takes place in the presence of a germination promoting agent, further preferably consisting of a compound belonging to the gibberellin family.

 According to an advantageous embodiment of the method, the germination step is preceded by a quenching step in an aqueous solution at room temperature or cold for one or more days, for example two days.

 According to yet another alternative embodiment of the process according to the invention, the step of extracting the SOD from the sprouted seeds comprises a grinding of the sprouted seed followed by an extraction in an aqueous buffered solution having a pH close to 8 at room temperature for a period of time sufficient to carry out SOD extraction, typically from a few tens of minutes to one or more hours, followed by filtration and recovery of the SOD-containing filtrate.

 According to an advantageous embodiment of the extraction process, further purification of the SOD can be carried out by treatment of the filtrate with a precipitating agent making it possible to eliminate the undesirable proteins, including proteases or lipoxygenase, and recovering the fraction not precipitated or constituting the supernatant which contains SOD.

 According to another even more advantageous embodiment, the non-precipitated fraction containing SOD can be dialyzed by dialysis against water or an aqueous solution, with a membrane preferably having a cut-off point of between 6000 and 8000 Dalton.

 According to yet another particularly preferred variant, a further purification of the dialyzed solution can be carried out by carrying out a purification on a chromatographic column which will preferably be constituted in the context of the invention by a QAE Sephadex column using an appropriate elution solution.

According to another advantageous embodiment of the process according to the invention, a stabilization of SOD obtained directly after the aforementioned extraction step, that is to say before a further purification, or after purification, can be carried out. addition of an H202 scavenger, such as a peroxidase with preferably a peroxidase cofactor also referred to as a peroxidase-specific reducing substrate. Preferably, the amount of peroxidase added to the
SOD expressed in a ratio of peroxidase unit relative to SOD units is between 10 and 400 according to the invention, whereas for the enzymatic cofactor of peroxidase it is preferably present in a concentration of between 0.001 M and 1M. compared to the SOD / peroxidase complex.

 According to another advantageous embodiment of the process according to the invention, it is also possible to stabilize the SOD by adding, after the extraction step, or after the purification step, at least one sugar, in particular a monosaccharide or a disaccharide and / or at least one polyol, in particular a polyol having an average molecular weight of between 50 and 1000 g / mol. Preferably, the sugar or the polyol will be added at a concentration of between 10 and 50% by weight of the SOD or of the final SOD complex, in the form of a crude or purified extract.

 After extraction without or with purification, an extract containing an SOD activity is obtained which can be determined by the method well known to those skilled in the art of Nebot C., et al., Published in Analytical Biochemistry, 214 ( 1993) pp. 442-451.

This method of Nebot is based on the property that any catalyst provided with SOD activity to accelerate, at alkaline pH, the autoxidation of a reagent R1 into a chromophore absorbing visible light, according to the following reaction:

Method S0> 525 also exploits a second reagent R2 which makes it possible to eliminate the major interferences due to the mercaptans which could be present in the sample to be analyzed, for example glutathione, with the aid of an alkylation reaction. very fast, depending on the reaction:

 The measurement of the SOD activity is carried out at a pH of 8.8, which allows an optimal sensitivity of the assay without inactivation of known natural SODs, such as, for example, copper-zinc, manganese or iron SODs.

 There is a dosing kit, marketed by Oxis International SA, (94385 BONNEUIVMARNE, FRANCE), for performing a spectrophotometric assay of SOD activity by this method SOD-525. This kit comprises two reagents, R1 and R2, and a buffer solution, buffer 3, which are respectively: King: a solution of chromogenic reagent R1 in 3.2 × 10 -2 M HC1 of the aforementioned formula; R2: a solution of the R2 mercaptan scavenger reagent in DMSO containing 25% (w / v) ethylene glycol; Buffer 3: a buffer at pH = 8.8 (at 37 ° C) containing 0.11 mM diethylenetriaminepentaacetic acid (DTPA).

With this kit from Oxis International, marketed under the name kit
SOD-525, the spectrophotometric measurements are carried out in glass vats of 1 cm optical path at a wavelength of 525 nm. The kinetic measurement of the evolution of the absorbance is carried out for 1 minute at 37 ° C.

 For each measurement, the reaction rate is determined by evaluating the maximum slope of the curve obtained. This slope corresponds to an autoxidation phase of R1. The results are expressed in absorbance units per minute.

The calculation of the enzymatic activity is carried out as follows:
Let Vc and Vs be the reaction rates corresponding respectively to the control and to the sample. The SOD activity of the sample to be analyzed is determined by calculating the correspondence between the experimental ratio Vs / Vc and the SOD activity, deduced from the following equation:
(1) Vs [SOD] with a = 0.073 and b = 0.93
Vc a [SOD] + b
A unit of activity SOD-525 defined by Oxis corresponds to a ratio
Vs / Vc equal to 2 under the conditions above.

 The value obtained is then multiplied by the dilution factor of the sample (factor 25) in the assay procedure. The results are expressed in units of SOD-525 activity per ml of sample.

In the context of the invention, the SOD enzymatic activity is measured after dilution of the SOD solution obtained so as to remain in Vs / Vc ratio values making it possible to establish a correct correspondence with the activity.
SOD.

 In the context of the invention, the solution of SOD obtained will generally be diluted, depending on the degree of purification resulting from the extraction process to a factor of 300, in order to obtain a ratio Vs / Vc of between 1 and 2.

 The activity of the SOD solution is determined by taking into account the dilution factor of the sample of the assay procedure (factor 25) and the dilution factor of the SOD solution itself (for example up to one factor of 300).

 The dosage examples will be given in connection with the examples.

 The invention also covers compositions in particular with free antiradical activity, for example cosmetic, pharmaceutical or agrifood compositions characterized in that they comprise, as one of the active ingredients, a vegetable SOD obtained from seeds of plants having undergone a stage germination.

 In this third aspect, the proportion of incorporation is variable and will depend on the intended application. Generally, the proportion of SOD incorporation will be from 0.01 to 30% by weight, more preferably from 0.1 to 10% by weight and more preferably of the order of 1 to 5%.

 According to an advantageous embodiment of this composition, the SOD is present in the form of SOD / peroxidase complex with preferably a peroxidase cofactor also called peroxidase-specific reducing substrate.

 Peroxidases catalyze the destruction of hydrogen peroxide (H2O2) produced during the SOD catalyzed disproportionation reaction of the superoxide anion in hydrogen peroxide as recalled at the beginning of the description in the presence of a specific reducing substrate called peroxidase cofactor. .

 Peroxidases are numerous and well known to those skilled in the art.

They are currently extracted from the spinal cord or myelo peroxidase, milk or lactoperoxidase, bovine or possibly human red blood cells or glutathione peroxidase or preferably according to the invention from black radish or horseradish peroxidase.

 Moreover, the specific reducing substrates of peroxidases are well known to those skilled in the art and are preferably chosen from glutathione, phenol, guaiacol, pyrogallol, mesitol, 3,5-dichloro-2-hydroxybenzene sulfonic acid (DCHBS ), aniline, p-toluidine, o-phenylene diamine, mesidine, ascorbic acid, dihydroxymaleic acid, cytochrome C, iodides, uric acid, phenolphthalein, 2,2'-azido-di (3-ethylbenzo-thiazoline (6) sulfonic acid ) (ABTS) and compounds such as SCN-, Cl-, Br- or I-.

 In the context of the invention, and in particular for an application in cosmetics or in pharmacy or foodstuffs, it is preferable to produce a SOD complex with a plant peroxidase extracted preferably from black radish, advantageously accompanied by its cofactor, preferably constituted by uric acid, ascorbic acid or dihydroxymaleic acid.

 The amount of peroxidase added to SOD is a function of the level of hydrogen peroxide released by SOD, and therefore of its enzymatic activity. The number of peroxidase units to be added to the SOD solution is expressed in a ratio of peroxidase units relative to the SOD units included according to the invention between 10 and 400.

 The enzymatic cofactor of peroxidase as previously described, which may be preferably uric acid, is added to the enzyme complex at a concentration of between 0.001 M and 1 M relative to the SOD / peroxidase complex.

 According to one preferred embodiment of the invention, the SOD or the SOD / peroxidase / peroxidase cofactor complex may be further stabilized by at least one sugar and / or at least one polyol added at a concentration of between 10 and 50% by weight. weight of SOD or final complex, in the form of a crude extract or in purified form.

 As sugar, use will in particular be made of a monosaccharide or a disaccharide and in particular trehalose and, as polyol, use will in particular be made of a polyol having an average molecular weight of between 50 and 1000 g / mol, for example glycerol, sorbitol, maltitol and mannitol. The addition of a sugar or a polyol makes it possible to stabilize in a particularly unexpected manner the activity of the SOD.

 Furthermore, according to yet another preferred embodiment of the invention, an antioxidant agent is advantageously lipophilic, preferably from the family of tocopherols and their derivatives, such as their esters such as acetates, linoleates or even phosphates because In particular, it has been unexpectedly discovered that such an antioxidant provides increased stability to SOD. Tocopherol phosphates are, for example, described in U.S. Patent Nos. 387,579 to LVMH RESEARCH. This result is particularly unexpected since the stability of the SOD enzyme is not related to oxidation problems and therefore the mechanism of action is currently unknown. Since this antioxidant is desirably lipophilic, it will be added to the composition in an oily phase, i.e. generally in the context of emulsion formation. The concentration of antioxidant agent will generally be from 0.01 to 3%, preferably from 0.1 to 1%, by weight relative to the total weight of the final composition.

 Other aims, features and advantages of the invention will become clear in the light of the explanatory description which follows, made with reference to several examples of embodiments of the invention given merely by way of illustration and which therefore can not in any way be understood. limit the scope of the invention. In the examples, all proportions are by weight unless otherwise indicated.

 The examples are an integral part of the invention and any feature of the examples which would appear novel with respect to any state of the art constitutes a general feature of the claimed invention as such.

Example 1
Extraction of SOD from barley germ
This extraction of SOD takes place as follows: a) Germination of barley:
Commercially available barley is used, for example the variety of spring barley Dallas which is soaked in an aqueous solution, for example tap water or preferably demineralized water, cold, for example at 15-C, for one or more days, for example two days.

 After this soaking, which is intended to swell the grains, and thus to prepare the germination, the actual germination step is carried out for several days, for example five days, preferably in the presence of a propellant. germination, such as gibberellic acid at a concentration of 0.1 mg / kg starting grains.

b) Extraction protocol:
The extraction protocol from sprouted barley, also called malt, is as follows:
After germination of the barley seeds, the malt is milled and then extracted in an aqueous buffer solution having a pH of about 8, which is preferably constituted by 50 mM Tris HCl + 1 mM EDTA, for about 1 hour at room temperature.

The extract is then filtered to remove the bark, then centrifuged at 4000G for 20 minutes to remove the polysaccharide portion and the filtrate or supernatant on which the SOD activity is determined by the above method of Nebot C., et al. (1993) using a commercial kit from Oxis
International, 94385 BONNEUIUMARNE, FRANCE. We get an activity
SOD-525 / g starting dry matter.

The SOD activity is measured after dilution of 1 ml of the SOD extract in 14 ml of water. 40 ul of the diluted solution are dosed. The measured Vs / Vc ratio is 1.95. According to equation (1) above, page 9, the SOD activity is 0.95 units
SOD-525. The SOD activity is therefore equal to 0.95 × 25 × 15 = 356.25 SOD525 / ml units, ie in this example 1482 SOD-525 units / g of starting dry matter.

(c) Complementary punishment:
The filtrate or supernatant obtained at the end of the centrifugation above is subjected to precipitation with a precipitating agent eliminating undesirable proteins such as proteases or lipoxygenase, for example ammonium sulphate at a concentration of 390 g. / l.

 Centrifugation at 4000 G for 20 minutes at 20C to remove the precipitate and recover the liquid fraction consisting of the supernatant containing the SOD.

 The supernatant can be advantageously dialysed to remove small molecules and salts present in high concentration having a molecular weight of less than 6000 Dalton. This dialysis is carried out against demineralized water with membranes whose cut-off point is 60008000 Dalton, for example 6000 Dalton. Indeed, SOD has a molecular weight of the order of 30000, which does not pass into the pores of such membranes, which allows to increase the specific activity of the enzyme.

 The SOD activity measured on the dialysate according to the preceding method is 7500 OD.sub.50-525 units / g of starting dry matter.

(d) Complementary possible supplemental column.

 From the dialysate obtained in step c) above, it is still possible to carry out a much more thorough purification by working on a chromatographic column.

 We can, by

Example 4
Extraction of SOD from pea germs
Commercially available white peas (Pisum sativum) are used.

The same protocol as that described in Example 1 is used and the SOD activity is measured on the product obtained in step b. We get an activity
SOD of 80 UISOdg starting dry matter.

Example 5
Measurement of the variation of the SOD activity at different germination times or with different barley varieties in the presence or absence of a seed germ activating agent such as a molecule derived from the gibberellin family Example 5-a
Measurement of the variation of the SOD activity at different germination times
The SOD activity was measured from the Dallas variety sprouted barley by varying the germination time and respecting the other general conditions of the germination protocol of Example 1-a.

 The results are shown in Table I below.

TABLE I

<tb> Activity <SEP> SOD <SEP> in <SEP> UISOdg <SEP> Time <SEP> of <SEP> germination <SEP> (day) <SEP>
<tb><SEP> of <SEP> dry matter <SEP>
<tb><SEP> N / A <SEP> 1 <SEP>
<tb><SEP> 120 <SEP> 2
<tb><SEP> 290 <SEP> 3
<tb><SEP> 410 <SEP> 4
<tb><SEP> 480 <SEP> 5
<Tb>
It can be seen from the results of Table I that the SOD activity is more than doubled after 1 day of germination and that it is again more than doubled on the third day of germination and that it considerably increases the fourth day. and fifth day.

Example 5-b
Measuring the influence of barley variety on SOD activity
The influence of the barley variety on the SOD activity was measured and the results obtained are reported in Table II below. This comparison was carried out at a constant germination time of 5 days but without a germination-promoting agent, the other conditions of the germination protocol being identical to that of the 1-a exemption.

TABLE II

<tb><SEP> variety of barley <SEP> Activity <SEP> SOD <SEP> in <SEP> UISOD / g
<tb><SEP> of <SEP> dry matter <SEP>
<tb><SEP> Natasha <SEP> 239
<tb><SEP> Felicie <SEP> 271
<tb><SEP> Dallas <SEP> 214
<tb><SEP> Magic <SEP> 218 <SEP>
<tb><SEP> Puffin <SEP> 247
<Tb>
It can be seen from Table II that there is a slight variation in the results obtained in SOD activity for various varieties of barley but that these results are perfectly consistent, which proves the reproducibility of the germination procedure according to the present invention.

Examples-c
Influence of the presence of molecule from the gbberellme family
The influence of the presence of a germinating agent, such as gibberellic acid, has also been determined and the results are shown in Table m. These tests were carried out with spring barley variety Natasha according to the protocol of Example 1 with a germination time of 5 days.

TABT; FÂIIm

<tb><SEP><SEP> Gibberellic acid <SEP> Activity <SEP> SOD <SEP> in <SEP> UISOD / g <SEP>
<tb><SEP> of <SEP> dry matter <SEP>
<tb><SEP> without <SEP> 239
<tb> with <SEP> 0.1 <SEP> mgSkg <SEP> of <SEP> grains <SEP> 407
<Tb>
It can be seen from Table 1 that the presence of a germination promoting agent makes it possible to increase the amount of SOD produced in a particularly unexpected manner.

example6
Stabilization of SOD by a polyol
SOD in the form of crude extract obtained in Example 1, step b) retains 60% of its enzymatic activity after 46 days at 20 ° C.

 It has been found that if 20% by weight of crude extract is added to the crude extract, that is to say SOD extract of step 1-b plus 20% sorbitol by weight of the final solution, the stability of the SOD enzyme is unexpectedly improved since 76% of the initial activity is found after 46 days at 20 ° C.

Example 7
Stabilization of SOD with sugar
It has been found that the enzymatic activity of the
SOD by adding a sugar, in particular a monosaccharide or a disaccharide.

In this example, we will use trehalose.

 If one proceeds as described in Example 6, but adding 30% by weight of trehalose to the crude extract of SOD obtained in Example 1 step b), relative to the weight of the final solution, it can be seen that It retains 75% initial activity after 46 days at 20 ° C, which is a remarkable improvement in stability.

Example 8
Stabilization of SOD by a peroxidase and a cofactor
The concentrated SOD solution obtained in step 1-d or obtained in an earlier step of Example 1 is complexed or combined with another enzyme capable of destroying the oxygenated water formed.

 The enzymes that can be used in this sense can be a catalase that converts H2 2 into H20 and O2 but catalase is not used as it is included in the list of unauthorized products in cosmetology. It is also possible to use a peroxidase which also catalyzes the destruction of H2 2 which nevertheless requires the presence of a specific reducing substrate or cofactor. The lists of peroxidases and cofactors have been given in the introduction of the description.

 In the context of this example, a plant peroxidase extracted from black radish or horseradish peroxidase, hereafter abbreviated as HRP, combined with an enzymatic cofactor constituted by uric acid, will be used as peroxidase.

In the context of this example, the amount of peroxidase added to the
SOD is about 10 peroxidase units per 400 SOD units, the SOD unit being measured according to the method previously described and the peroxidase unit being determined by the method described by Bergmeyer HU in Methods of Enzymatic Analysis (1974), vol. 1, 2nd ed. page 494.

 The enzymatic cofactor of the peroxidase here constituted by uric acid is added to the SOD / peroxidase enzyme complex in a concentration of between 0.01 and 1 M, and preferably 0.5 M.

 Preferably, the complex thus formed is further stabilized by the addition of added sugar or polyol at a concentration selected in this example to 30% by weight of the final solution.

 This SOD / peroxidase / cofactor complex can be used as it is to formulate compositions with antiradical or free radical scavenging activity, be they cosmetic, pharmaceutical or agri-food or other compositions. This complex is also the subject of stability tests, free radical capacity and toxicology object respectively of Examples 9, 10 and 11 below.

example9
Stability tests
The stability of the enzymatic activity of the complex formed in Example 8 was carried out at 20 ° C. and 45 ° C. respectively and the results obtained are listed in Table IV below.

TABLE IV

<tb> Days <SEP> Activity <SEP> SOD <SEP> Activity <SEP> SOD
<tb><SEP> Units <SEP> SOD-525 / ml <SEP> Units <SEP> SOD-525 / ml
<tb><SEP> 2o.C <SEP> 4 <SEP>
<tb><SEP> 0 <SEP> 5025 <SEP> 5025
<tb><SEP> 7 <SEP> 4800 <SEP> 4275
<tb><SEP> 11 <SEP> 7575 <SEP> 3575
<tb><SEP> 13 <SEP> 5925 <SEP> 3150
<tb><SEP> 18 <SEP> 4500 <SEP> 2800
<tb><SEP> 20 <SEP> 2475 <SEP> 2400
<tb><SEP> 35 <SEP> 3975 <SEP> 2475
<Tb>
Table IV shows that enzyme activity is stable at 20.degree. C. for 1 month, since at 35 days 80% of the enzymatic activity is retained, while at 45.degree. 50%, which represents a remarkably low activity drop for an enzyme at this temperature.

 It is thus observed that this plant / peroxidase / cofactor SOD complex has a remarkable stability, particularly at 45.degree. C., which is unexpected for a person skilled in the art.

Example 10
Measurement of Free Radical Activity or Free Radical Scavenging Three methods of in vitro evaluation of radical scavenging have been used: (a) the first method uses an enzymatic system consisting of xanthine oxidase which, by oxidizing its substrate, xanthine, generates superoxide radicals. The latter are capable of reducing cytochrome C (Fe3 +) to cytochrome C (Fe2 +), a reaction whose kinetics can be followed by UV-visible spectrophotometry at 550 nm. ns are also the substrate radicals of SOD which can thus compete with cytochrome C for the capture and removal of superoxide radicals. By adding a preparation containing SOD activity, this reduction kinetics is slowed down. Calculation of the SOD activity is performed by calculating the percent inhibition of cytochrome C reduction by the SOD tested.

 An SOD unit expressed in the international system (1 UlSOD) corresponds to the activity required to reduce by 50% the cytochrome C reduction rate, at a temperature of 25-C and a pH of 7.8.

 The test sample is diluted to determine the conditions under which 50% inhibition of cytochrome C reduction, corresponding to 1 international system unit, is achieved.

 The total SOD activity of the test sample is then determined by reducing the sample volume dosed to the total sample volume and taking into account the initial dilution factor of the sample.

 The SOD activity of the complex formed in Example 8 determined by this method is 4455 UlsoD units / ml.

b) The second method consists of a kit sold commercially by OXIS INTERNATIONAL, under the trade name kit SOD-525, based on the property that any catalyst provided with SOD activity, to accelerate the oxidation of a tetracyclic catechol derivative (5, 6, 6a, 11b-tetrahydro-3,9,10-trihydroxybenzofluorene).

 This assay method makes it possible to measure the SOD activity in the form of SOD-525 unit which is defined as the quantity of dismutase which multiplies by 2 the oxidation rate of the fluorinated derivative.

The SOD activity of the complex formed in Example 8, measured by this method, is 4375 units SOD-525 / ml
It is thus noted that the SOD activity values obtained by the two methods are essentially identical.

c) The third method used is the Paramagnetic Resonance of the electron or abbreviated RPE.

EPR is a technique for characterizing the spin states of single electrons of molecules, more particularly used to characterize free radicals. Evaluation of the antiradical activity of the complex
SOD / peroxidase / cofactor obtained in Example 8 by RPE, makes it possible to distinguish the trapping activity of superoxide radicals and hydroxyl radicals (Rosen
GM and Rauckman EJ in Methods In Enzymology (1984), 105, pages 198209).

 The very short life time of stamp radicals, about 10-11 seconds for OH-, limits the detection capacity of the latter. For this reason, the radicals 2 and OH 'are highlighted by a probe molecule called spin-trap. This molecule resonates in the presence of free radicals and stabilizes the latter in the form of complexes having a characteristic spectrum detectable in EPR.

 The scavenging effect of a product under test is directly evidenced by a decrease in the intensity of the signal emitted by the spin-trap molecule.

 EPR measurements are performed with a Brucker ESP 106 spectrometer at room temperature; the spin trap used is 5,5-dimethyl-1-pyrroline-1-oxide (DMPO).

 The superoxide anion is produced by the enzymatic xanthine (1.5 mM) / xanthine oxidase system (12 mU / ml), in the presence of DMPO (80 mM) and 50% ethanol (v / v). The SOD / peroxidase / cofactor complex obtained in Example 8 is dissolved in ultrapure water at various concentrations.

 The hydroxyl radical is produced by photolysis (UVB) of hydrogen peroxide at 0.2% (v / v) in aqueous solution, in the presence of DMPO (160 mM).

The SOD / peroxidase / cofactor complex obtained in Example 8 is dissolved in ultrapure water at various concentrations.

 The intensities of the RPE signal are calculated by double integration of the lowest field line. The percentages of protection of the product under test are obtained from the values of the controls without test product.

% protection = - Spreads - Stemoin | x 100
So - Stemoin
Sproduit: integration of the RPE signal obtained with the product under test or the reference
Stemoin: integration of the RPE signal obtained with the witness
So: integration of the RPE signal obtained in the absence of free radicals
Effect on superoxide radical
The SOD / peroxidase / cofactor complex obtained in Example 8 decreases the RPE signal of the superoxide anion in a dose-dependent manner. AS and 10% (v / v), the complex inhibits the RPE signal at 44% and 54%.

Effect on hydroxyl radical
The SOD / peroxidase / cofactor complex obtained in Example 8 decreases the RPE signal of the hydroxyl radical in a dose-dependent manner. At the concentration of 0.3% (v / v), the effect is spectacular since it inhibits the RPE signal to 93%.

 The SOD / peroxidase / cofactor complex obtained in Example 8 has very good antiradical effects with respect to the superoxide anion and the hydroxyl radical even at low concentrations, generally used in cosmetic formulations.

 2. A method of ex vivo evaluation of the antiradical activity on normal human fibroblasts in culture was used.

 After irradiation with UVA, the free radicals formed in a culture of human fibroblasts are quantified by an appropriate method. The use of antiradical agents makes it possible to reduce the oxidative stress, and the calculation of the efficacy of the plant SOD / peroxidase / cofactor complex according to Example 8 at different concentrations is carried out.

 Normal human fibroblasts are incubated for 1 hour in the presence of the plant SOD / peroxidase / cofactor complex according to Example 8, tested at 0.01, 1 and 10% (v / v) in demineralized water. The cells are subjected to a radical stress caused by UVA light radiation (10 J / cm 2). The free radicals formed (hydroperoxides) are detected by means of a suitable probe, which is converted in the presence of hydroperoxides, into a quantifiable fluorescent derivative.

The results are first expressed in arbitrary fluorescence unit per culture well. Then an efficiency is calculated by applying the following formula:
E (%) = [(; TI-IF) / (Fl-FNI)] x (-100) where:
E: Percentage Efficacy FNI: Fluorescence Observed with Non-Irradiated Fibroblasts
Fl: Fluorescence observed with Irradiated Fibroblasts Ffl: Fluorescence observed with Treated and Irradiated Fibroblasts
An aqueous solution containing 1% of plant SOD / peroxidase / cofactor complex formed according to the invention is capable of reducing by more than 80% the harmful effects linked to an oxidative stress generated by UVA.

 This efficiency is greater than that obtained with a reference mixture glutathione / vitamin C, however, known to be particularly effective, and which also has enormous stability problems.

 At the lowest concentrations of use, the plant SOD / peroxidase / cofactor complex according to Example 8 thus allows effective protection of human fibroblasts cultured after UVA oxidative stress.

 * 3. A method of in vivo evaluation of the antiradical activity on cutaneous tissues of healthy volunteers was used.

 Free radicals formed after UVA irradiation peroxidize unsaturated lipids in skin tissues. Stripping makes it possible to collect the corneocytes with the peroxidized entities. In the reaction medium, the peroxides are revealed by means of a fluorescent probe which emits a fluorescence directly proportional to the level of peroxides present.

 Seven female volunteers, aged between 20 and 38 years old, with a type II or III phototype, that is to say with normal Caucasian skin are selected.

 Three zones of the forearm are demarcated and two of these zones are irradiated with UVA (10 J / cm2) using a Sun 3000 St UVA lamp). The fugitive red trace obtained is not visible the day after irradiation and no inflammatory process is observed.

 An aqueous solution containing 1% of vegetable SOD complex / peroxidase / cofactor formed according to the invention is applied 4 times at 2 hours of interval (2, uVcm2).

 24 hours after irradiation with UVA, a sample of the stratum corneum is made by 2 successive stripping.

 The peroxides formed are revealed on the second stripping using a fluorescent probe which gives an overall response to the peroxidation process.

The results are first expressed in arbitrary fluorescence unit. Then an efficiency is calculated by applying the following formula:
E (%) = [(ZIT - Z1) / (ZI - ZNI)] x (-100) where:
E (): Efficiency in percent Z1T: Fluorescence measured in the Irradiated Zone Treated ZI: Fluorescence measured in the Irradiated Zone ZNI: Fluorescence measured in the Non-Irradiated Zone.

 An aqueous solution containing 1% of plant SOD / peroxidase / cofactor complex formed according to the invention is capable of protecting with an effectiveness of 65%, cutaneous radical stress induced by a UVA irradiation.

Example 11
Toxicology
The toxicology tests were carried out on the complex according to the invention obtained in Example 8 on the evaluation of the primary cutaneous irritation in the rabbit (9a), on the evaluation of eye irritation in the rabbit ( 9b), the absence of abnormal toxicity by single oral administration in the rat (9c) and by the study of the sensitizing power on the guinea pig (9d).

Evaluation of primary cutaneous irritation in rabbits
The preparation obtained in Example 8 was applied without dilution to the dose of 0.5 ml on the skin of 3 rabbits according to the method recommended by the directive
OECD n404 concerning the study of "acute irritant / corrosive effect on the skin".

 The results of this test led to the conclusion that the preparation obtained in Example 8 according to the invention can be considered as not irritating to the skin even in the undiluted state.

11b- Evolution of ocular irritation in the tapin
The same preparation obtained in Example 8 was instilled pure in one go, at a rate of 0.1 ml, in the eye of 3 rabbits according to the method recommended by the OECD guideline No. 405 of 24 February 1987 concerning the study of "acute irritant / corrosive effect on the eyes".

 The results of this test make it possible to conclude that the preparation obtained in Example 8 according to the invention can be considered as not irritating to the eyes within the meaning of Directive 91/326 EEC used pure or without dilution.

llc- Test on the absence of abnormal toxicity by single oral administration in the State
The preparation obtained according to Example 8 according to the invention was administered once orally at a dose of 5 g / kg of body weight, to 5 male and 5 female rats according to a protocol inspired by the WHO guideline. OECD N401 of 24 February 1987 and adapted to cosmetic products.

 The results of this test make it possible to conclude that, under these experimental conditions, the preparation obtained in Example 8 according to the invention does not exhibit any abnormal toxicity.

- Study of sensitizing power in cobalt
The SOD complex solution obtained in Example 8 was the subject of a sensitization power search according to the Magnusson method and
Kligman published in J. invest. Derm. (1969), 52, pp. 268-276. This solution of SOD complex was applied as such to the skin of 35 guinea pigs previously treated with Freund's adjuvant and divided into 2 experimental batches, respectively control group and treated batch.

 No reaction was noted in both experimental batches.

 The results obtained show that the vegetable SOD complex / peroxidase / cofactor formed according to the invention does not cause the appearance of any sensitization reaction.

Example 12
Preparation of a Composition in the Form of a Cosmetic Emulsion Containing the Vegetable SOD / Peroxidase SOD Preparation According to the Invention
The preparation obtained in Example 8 according to the invention is incorporated in a cream at a concentration of 5% by weight relative to the total weight of this cream.

This cream has the following composition:
INCI name:
A- Steareth-2 ...... 3%
Steareth-21 ..... ...... 2%
Polypropylene Glycol-15 Stearylether ....... 9%
Cetearyl alcohot. . . ...... 2,5%
B-Butylene glycol ...... 4.5%
Water. ...... 73.3%
C- Preservative comprising parabens and phenoxyethanol ..... 0.5%
D-Tocopherol ..... 0.2%
E-Complex
SOD plant / peroxidase / cofactor according to the invention of Example 8 .... 5%
The enzymatic activity in the cream is measured after separation of the aqueous and oily phases, carried out under specific conditions: 2 g of cream are diluted 5 times in 8 g of 0.2 M Tris (hydroxymethyl) aminomethane buffer, pH 8, 5, then 10% NaCl are added. The mixture is subjected to violent agitation (of the type obtained with the aid of an Ultra-turax) for 5 minutes and then centrifuged at 5000 rpm for 25 minutes. The enzymatic activity is measured directly in the extracted aqueous phase.

 The stability of the enzymatic activity of the preparation obtained in Example 8 incorporated into a cream, described above, was studied at 20 ° C and 45 ° C for 40 days.

 The results are listed in Table V.

TABLE V

<tb><SEP><SEP> SOD <SEP> activity of <SEP><SEP> sOD <SEP> activity of <SEP><SEP> SOD <SEP> activity of
<tb><SEP> complex <SEP> SOD <SEP> complex <SEP> SOD <SEP> the raw <SEP> SOD <SEP>
<tb><SEP> stabilized <SEP> according to <SEP> stabilized <SEP> according to <SEP> of extraction <SEP>
<tb> Days <SEP> the invention <SEP> the invention <SEP> obtained <SEP> to
<tb><SEP> obtained <SEP> at <SEP> obtained <SEP> at <SEP> exempts <SEP> 1 <SEP> step
<tb><SEP> the free <SEP> 8 <SEP> example <SEP> 8 <SEP> b) <SEP> (no
<tb><SEP> (%) <SEP> (%) <SEP><SEP> stabilized) <SEP> (%)
<tb><SEP>20'C<SEP> 45C <SEP> 45C <SEP>
<tb><SEP> 0 <SEP> 100 <SEP> 100 <SEP> 100
<tb><SEP> 1 <SEP> 100 <SEP> 100 <SEP> 0
<tb><SEP> 3 <SEP> 89 <SEP> 83 <SEP> 0
<tb><SEP> 6 <SEP> 69 <SEP> 83 <SEP> 0
<tb><SEP> 18 <SEP> 92 <SEP> 64 <SEP> 0
<tb><SEP> 25 <SEP> 70 <SEP> 66 <SEP> 0
<tb><SEP> 40 <SEP> 60 <SEP> 50 <SEP> 0
<Tb>
The enzymatic activity found in the cream after 40 days at 20 ° C. is 60% for the stabilized SOD form. More remarkable still the enzymatic activity found in the cream stored for 40 days at 45C is 50% while the unstabilized form does not withstand the temperature.

 Stability studies of the complex according to the invention, incorporated in a cream, show that the complex has a remarkable stability at 20 ° C and particularly unexpectedly at 45in.

Example 13
Preparation of plant SOD complex with various peroxidases
Example 13A
SOD complex / horseradish peroxidase
The SOD solution obtained in step 1c of Example 1 in which a number of units of horseradish plant peroxidase, called
HRP, in a ratio of HRP units to SOD units between 10 and 400.

 The vegetable SOD preparation / horseradish peroxidase thus obtained can be used as it is or preferably combined with an enzymatic cofactor, for example uric acid or ascorbic acid or dihydroxymaleic acid.

Example 13B
SOD vegetate / peroxidase complex of Arthromyces ramosus
The procedure is as in Example 13A except that it uses a peroxidase obtained from Arthromyces ramosus commercially available, for example under the reference Sigma P 4794.

Example 13C
Plant SOD Complex / Soy Peroxidase
The procedure is as described in Example 13A except that a peroxidase extracted from soybeans commercially available under the reference Sigma P 1462 is used.

Example 14
Stabilization of plant SOD / peroxidase / cofactor complex with a sugar or oolvol
Stabilization of the plant SOD / peroxidase / cofactor enzyme complex obtained in Example 8 is improved by adding at least one sugar or polyol according to the following embodiments:
Example 14A
Addition of, lverterol
To the preparation obtained in Example 8 according to the invention, the glycerol is added to a concentration chosen in this example to 30% by weight of the final solution, with stirring at room temperature.

.Example 14B
Addition of sorbitol
The procedure is as in Example 14A, except that sorbitol is used.

Example 14C
Adding maltitol
The procedure is as in Example 14A, except that maltitol is used.

Exemole 14D
Adding mannitol
The procedure is as in Example 14A, except that mannitol is used.

Example 14E
Addition of trehalose
The procedure is as in Example 14A, except that trehalose is used.

Example 15
Enzymatic complex formation with different peroxidase cofactors
The procedure is as described in Example 8, except that peroxidase cofactors other than uric acid, which was used in Example 8, are used.

Example 15A
Ascorbic acid
The procedure is as described in Example 8, if

Exempted 16
Cosmetic composition of oil-in-water emulsion type, more particularly suitable for antiradical cosmetic preparations. anti-age.

anti-wrinkle and anti-stress skin
A composition in the form of an oil-in-water emulsion is conventionally prepared from the following five fractions A, B, C, D and E having the percentage composition indicated:
INCI name:
A-Oily phase comprising:
Steareth-2 .. .... 3%
Steareth-21 .... . ...... 2%
Polypropylene glycol-15 stearyl ether ...... 9%
Cetearyl alcohol. . . ...... 2,5%
B-Butylene glycol ... ... 4.5%
Water. . .... . ........... 73.3%
C- Preservative comprising parabens and phenoxyethanol ...... 0.5%
D-Tocopherol ...... 0.2%
E-plant SOD complex / peroxidase / cofactor according to the invention of the exemption 8 ............ 5 8 5%
After heating the fatty and aqueous phases at 80 ° C. separately, phase B is mixed in phase A, followed by phase C, phase D and phase E until an oil-in-water emulsion is obtained.

Example 17
Pharmaceutical composition with anti-inflammatory activity
This pharmaceutical composition comprises, in addition to a conventional pharmaceutical active ingredient, a preparation as obtained in Example 8 according to the invention, at a rate of 5% by weight, in admixture with a pharmaceutically acceptable excipient.

Exempted 18
Agmairmental composition stabilized against oxidation
This agri-food composition has a stability with respect to rancidity and comprises, in addition to the conventional active agrifood ingredients, 5% by weight of preparation obtained according to the invention 8 which are incorporated at the same time as the other active ingredients .

Claims (26)

 1. Use of plant seeds after germination as a source of superoxide dismutase or SOD extraction.  2. Use according to claim 1, characterized in that the seeds of plants are cereal seeds or seeds of leguminous plants or seeds of oleaginous plants.  3.Use according to claim 2, characterized in that the cereals comprise wheat or barley, preferably barley (spring or winter varieties) and the legumes include lentil or pea seeds and oilseeds include soybeans.  4. Use according to one of the preceding claims, characterized in that the germination step comprises germination for a period of time sufficient to increase the SOD activity.  5. Use according to claim 4, characterized in that the germination period is carried out in water, preferably in the presence of a germination promoting agent, at room temperature.  6. Use according to claim 5, characterized in that the agent promoting germination is constituted by a molecule from the family of gibberellins.  7. Use according to one of the preceding claims, characterized in that the germination of the seed is preceded by soaking in an aqueous solution at room temperature or cold for one or more days.  8. A process for extracting superoxide dismutase or SOD, characterized in that the source of seeds of plants having undergone a germination step, in particular cereal seeds or seeds of leguminous plants or seeds of oleaginous plants.  9. Method according to claim 8, characterized in that the germination step comprises suspending the seeds of plants in an aqueous solution for several days at room temperature.  10. The method of claim 8 or 9, characterized in that the germination step takes place in the presence of a germinating agent, further preferably consisting of gibberellic acid.  11. Method according to one of claims 8 to 10, characterized in that the germination step is preceded by a quenching step in an aqueous solution at room temperature or cold for one or more days.  12. Method according to one of claims 8 to 11, characterized in that the step of extracting the SOD from the sprouted seeds comprises grinding the sprouted seed followed by extraction in an aqueous buffered solution having a pH close to 8 at room temperature for a period of time sufficient to carry out the extraction of SOD, generally from a few tens of minutes to one or more hours, followed by filtration and recovery of the filtrate containing SOD.  13. Process according to claim 12, characterized in that a further purification of the SOD is carried out by treatment of the filtrate with a precipitating agent making it possible to eliminate undesirable purities, including proteases or lipoxygenase, and recovery of the fraction not precipitated or constituting the supernatant which contains SOD.  14. The method of claim 13, characterized in that dialysis of the non-precipitated fraction containing SOD by dialysis against water, with a membrane preferably having a cutoff of between 6000 and 8000 daltons. .  15. The method of claim 14, characterized in that carries out a complementary purification of the dialyzed solution by proceeding by chromatographic column purification.  16. Method according to one of claims 8 to 15, characterized in that stabilizes the SOD with an H202 scavenger, such as a peroxidase with preferably a peroxidase cofactor also called peroxidase-specific reducing substrate.  17. Method according to one of claims 8 to 16, characterized in that stabilizes the SOD by adding at least one sugar, in particular a monosaccharide or a disaccharide, and / or at least one polyol in particular having a average molecular weight between 50 and 1000 g / mol.  18. Composition including free antiradical activity, for example cosmetic composition, pharmaceutical or food, characterized in that it comprises as one of the active ingredients a plant superoxide dismutase obtained from seeds of plants having undergone a germination step.  19. A composition according to claim 18, characterized in that the proportion of SOD is between 0.01 and 10% by weight, better still 0.1 to 10% by weight and more preferably of the order of 5%, under stabilized form in the presence of an H 2 O 2 scavenger, such as peroxidase.  20. A composition according to claim 18 or 19, characterized in that the superoxide dismutase is present in the form of SOD / peroxidase complex with preferably a peroxidase cofactor also called peroxidase-specific reducing substrate.  21. Composition according to claim 20, characterized in that the above-mentioned peroxidase is chosen from the group consisting of a spinal cord peroxidase or myelo peroxidase, a lactoperoxidase, a glutathione peroxidase or a horseradish peroxidase and the reducing substrate specific for peroxidase. is preferably selected from glutathione, phenol, guaiacol, pyrogallol, mesitol, 3,5-dichloro-2-hydroxybenzene sulfonic acid (DCHBS), aniline, p-toluidine, o-phenylene diamine, mesidine, ascorbic acid, dihydroxymaleic acid, cytochrome C, iodides, uric acid, phenolphthalein, 2,2'-azido-di (3-ethylbenzo-thiazoline (6) sulfonic acid (ABTS) and compounds such as SCN-, Cl-, Br- or I-.  22. Composition according to one of Claims 18 to 21, characterized in that the said composition is a cosmetic, pharmaceutical or food-processing composition and SOD stabilized with a plant peroxidase preferably extracted from black radish, accompanied by a peroxidase cofactor constituted by uric acid, ascorbic acid or dihydroxymaleic acid.  23. Composition according to one of claims 19 to 22, characterized in that the amount of peroxidase added to SOD, expressed in a ratio of peroxidase units relative to the SOD units included according to the invention between 10 and 400.  24. Composition according to one of claims 19 to 23, characterized in that the enzymatic cofactor of peroxidase is present in a concentration of between 0.001M and 1M relative to the complex SOD / peroxidase.  25. Composition according to one of Claims 19 to 24, characterized in that the SOD or the SOD / peroxidase complex with optionally peroxidase cofactor is stabilized by at least one sugar, in particular a monosaccharide or a disaccharide, and / or less than one polyol in particular having a molecular weight of between 50 and 1000 g / mol, especially at a concentration of between 10 and 50% by weight of SOD or of the complex of SOD, in the form of a crude extract or in purified form.  26. A composition according to one of claims 18 to 25, characterized in that an antioxidant agent is advantageously lipophilic, preferably of the family of tocopherols and their derivatives, in particular their esters such as acetates, linoleates or even the phosphates, especially at a concentration of 0.01 to 3% by weight, better still 0.1 to 1% by weight, relative to the total weight of the final composition.